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Background Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate freezing and liquid nitrogen storage have become “routine” practice in many cell processing facilities, there is clearly room for improvement, as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of DMSO that can contribute to the morbidity and mortality of the transplant therapy. Methods This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor™ (BioLife Solutions, Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 × 107 cells/1 ml sample vial were prepared for cryopreservation in the following solutions: 1) FHCRC standard – Normosol-R, 5% HSA, 10% DMSO, and 2) CryoStor™ CS10 (final diluted conc. of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of one week. Vials were then thawed and evaluated for TNC, Viability, CD34, and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose. Results The PBSC samples frozen in CryoStor™ CS10 yielded significantly improved post-thaw recoveries for total viable CD34+, CFU, and viable granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8 fold increased recovery of viable CD34+ cells (P = 0.005), a 1.5 fold increase in CFU-GM numbers (P = 0.030), and a 2.3 fold increase in granulocyte recovery (P = 0.045). Discussion This study indicates that use of CryoStor™ for cryopreservation can yield significantly improved recovery and in vitro functionality of the stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while also not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary, however these findings imply use of CryoStor™ for cryopreservation might ultimately result in improved engraftment for those patients with lower content of CD34+ cells in their PBSC collections, along with reducing the requirement for additional apheresis collections, and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.

An optimal cooling rate is one of the critical factors influencing the survival of cells during cryopreservation. In this paper we describe a novel device, named the box-in-box, which was developed for optimal cryopreservation of human hematopoietic stem cells (HSC). This work presents the design of the device, a mathematical formulation describing the expected temperature histories of samples during the freezing process, along with actual experimental results of thermal profile tests. In experiments, when the box-in-box device was transferred from room temperature to a −80 °C freezer, a cooling rate of −1~−3.5 °C/min, which has been widely used for the cryopreservation of HSC, was achieved. In order to further evaluate this device, HSC cryopreservation was compared between the box-in-box device and a commercially available controlled rate freezer (CryoMed). The experimental data, including total cell population and CD34+ hematopoietic progenitor cell recovery rates, viability, and cell culture colony assays, showed that box-in-box worked as well as CryoMed instrument. There was no significant difference in either survival rate or the culture/colony outcome between the two devices. In conclusion, the box-in-box device can work as a cheap, durable, reliable and maintenance-free instrument for the cryopreservation of HSC. This concept of a box-in-box may also be adapted to other cooling rates to support cryopreservation in a wide variety of tissues and cells.

The cooling process is critical for the cryopreservation of human hematopoietic stem cells (HSCs). Currently, programmed freezing methods and uncontrolled cooling methods are in use, both having obvious disadvantages. In this article, a novel device termed Box-in-Box (BIB) was developed and evaluated by in vitro cryopreservation tests in 2 different operation modes ("against-side" mode for Group I (n = 10), and "in-middle" mode for Group II (n = 10), respectively), and compared with an uncontrolled cooling method (Group III (n = 7), Styrofoam boxes) as well as a conventional programmed freezer method (Group IV (n = 10), CryoMed TM 1010, Cryogenic Tech., FL). Recorded temperature profiles of samples cryopreserved with BIB show that a consistent cooling procedure with a rate around -1°C to -3.5°C/min can be achieved during their transfer from room temperature to an -80°C freezer. Statistical analysis of the stem cell population recovery, survival, and colony generation recovery shows that there is no significant difference (P > 0.26) among the methods using the BIB or programmed freezer (Group I, Group II, and Group IV), and their related deviations are smaller than the uncontrolled cooling rate method (Group III). Methods using the BIB (Group I and Group II) generated significantly better cell survival rate (P < 0.01) than the uncontrolled cooling rate method (Group III). The results indicate that the controlled cooling rate methods (BIB or CryoMed PF) are more consistent and reliable for clinical use. Considering the advantages of low cost, durability, and no liquid nitrogen consumption for the cooling process, the BIB can be a good alternative to the programmed freezers for the cryopreservation of HSCs.